A focal adhesion factor directly linking intracellularly motile Listeria monocytogenes and Listeria ivanovii to the actin-based cytoskeleton of mammalian cells.

Abstract

The surface‐bound ActA polypeptide of the intracellular bacterial pathogen Listeria monocytogenes is the sole listerial factor needed for recruitment of host actin filaments by intracellularly motile bacteria. Here we report that following Listeria infection the host vasodilator‐stimulated phosphoprotein (VASP), a microfilament‐ and focal adhesion‐associated substrate of both the cAMP‐ and cGMP‐dependent protein kinases, accumulates on the surface of intracytoplasmic bacteria prior to the detection of F‐actin ‘clouds’. VASP remains associated with the surface of highly motile bacteria, where it is polarly located, juxtaposed between one extremity of the bacterial surface and the front of the actin comet tail. Since actin filament polymerization occurs only at the very front of the tail, VASP exhibits properties of a host protein required to promote actin polymerization. Purified VASP binds directly to the ActA polypeptide in vitro. A ligand‐overlay blot using purified radiolabelled VASP enabled us to identify the ActA homologue of the related intracellular motile pathogen, Listeria ivanovii, as a protein with a molecular mass of approximately 150 kDa. VASP also associates with actin filaments recruited by another intracellularly motile bacterial pathogen, Shigella flexneri. Hence, by the simple expedient of expressing surface‐bound attractor molecules, bacterial pathogens effectively harness cytoskeletal components to achieve intracellular movement.

Vasodilator-stimulated phosphoprotein restricts cell-to-cell spread of Shigella flexneri at the cell periphery

“…To test whether the observed VASP-and EVL-associated decreases in cell-to-cell spread were due to an effect on bacterial actin-based motility, we compared the efficiency of bacterial actin tail formation per se in the VASP, EVL and Mena cells with that in cells lacking all Ena/VASP proteins. Consistent with the possibility that these proteins might modulate this process, and as we and others have previously described for VASP and Mena (Ally et al, 2004;Chakraborty et al, 1995;Gouin et al, 1999), each Ena/VASP protein co-localized with the polymerized actin in S. flexneri actin tails (Fig. S2).…”

“…During L. monocytogenes infection, Ena/VASP proteins bind directly to the bacterial surface protein ActA, leading to recruitment of the actin nucleator Arp2/3, and modulating the speed and directionality of bacterial movement through the cell cytoplasm and into adjacent cells (Auerbuch et al, 2003;Chakraborty et al, 1995;Welch et al, 1997Welch et al, , 1998.…”

“…As for L. monocytogenes, Ena/VASP proteins localize to the Shigella actin tail (Ally et al, 2004;Chakraborty et al, 1995;Gouin et al, 1999). However, Mena is not required for actin tail assembly or intracellular motility of S. flexneri (Ally et al, 2004).…”

Analysis and Regulation of Vasodilator-stimulated Phosphoprotein Serine 239 Phosphorylation in Vitro and in Intact Cells Using a Phosphospecific Monoclonal Antibody

“…VASP colocalizes with profilins and binds directly to their poly(L-proline) binding site (18), binds to and colocalizes with zyxin and vinculin (16,19), and also directly binds to Listeria monocytogenes surface protein ActA, which is essential for the actin polymerization-based intracellular motility of this pathogen (20). Functional evidence indicates that VASP is a crucial factor involved in the enhancement of spatially confined actin filament formation (16,20,21).Three distinct phosphorylation sites were biochemically identified in VASP (serine 157, serine 239, and threonine 278) which are used in vitro and in intact human platelets by both cAPK and cGPK and by the serine/threonine protein phosphatases 2A and 2B with overlapping selectivity (8,22). Phosphorylation of serine 157, the site preferred by the cAPK, leads to a marked shift in apparent molecular mass of VASP in SDS-PAGE from 46 kDa to 50 kDa (6, 8).…”

“…VASP colocalizes with profilins and binds directly to their poly(L-proline) binding site (18), binds to and colocalizes with zyxin and vinculin (16,19), and also directly binds to Listeria monocytogenes surface protein ActA, which is essential for the actin polymerization-based intracellular motility of this pathogen (20). Functional evidence indicates that VASP is a crucial factor involved in the enhancement of spatially confined actin filament formation (16,20,21).…”